Estimation with proteins

The void volume can be estimated with a solution of Dextran Blue (5 mg/ml). The BSA preparation contains the monomeric as well as the dimeric form of the protein. 

The isotope dilution principle, first employed by Hevesy and Hobbie (133) in 1932 for the determination of lead in ores, was applied by Schoenheimer et al. (241) to the determination of amino acids. [Shemin and Foster (248) have reviewed this topic.] An N15-amino acid derivative was added to a protein hydrolyzate, a sample of the amino acid to be determined was isolated and purified, the excess N15 in this product was estimated with the mass spectrograph, and the grams of amino acid originally present were calculated from Equation 2. 

In the particular example shown, zinc sulfate and barium hydroxide are being dispensed into the test tube so as to precipitate the proteins. The filtrate obtained is the filtrate from 10 microliters of serum. This can be used for several purposes and in the application being referred to, an amount equivalent to 3 microliters is being used for sugar determination, by the hexokinase procedure and an amount equivalent to 3 microliters is being used for urea estimation with diacetylmonoxime (15). 

As described above, it will be normal to assume that the dose interval is 24 hours, i.e., once-a-day dosing. Absorption can be estimated with good confidence from absorption in the rat (see Section 6.1). Clearance is the sum of the predicted hepatic, renal, biliary and extrahepatic clearance. Hepatic clearance can be derived from in vitro studies with the appropriate human system, using either microsomes or hepatocytes. We prefer to use an approach based on that described by Houston and Carlile [83], Renal clearance can be predicted allometrically (see section 6.8.1). The other two potential methods of clearance are difficult to predict. To minimize the risks, animal studies can be used to select compounds that show little or no potential for clearance by these routes. As volume can be predicted from that measured in the dog, after correction for human and dog plasma protein binding (see Section 6.2), it is possible to make predictions for all of the important parameters necessary. 

In summary, there are three important generalizations about error estimation in protein crystallography. The first is that the level of information varies enormously as a function primarily of resolution, but also of sequence knowledge and extent of refinement. The second generalization is that no single item of information is completely immune from possible error. If the electron density map is available or indicators such as temperature factors are known from refinement, then it is possible to tell which parameters are most at risk. The third important generalization is that errors occur at a very low absolute rate 95% of the reported information is completely accurate, and it represents a detailed and objective storehouse of knowledge with which all other studies of proteins must be reconciled. 

The photometric estimation of protein concentration is subject to some special features Proteins interact with each other depending on their concentration and may change their secondary and/or tertiary structure in a concentration- dependent manner (especially denaturation in diluted solutions). These changes affect the absorption of light, i.e., concentration dependence of molar absorption coefficient e therefore, the Beer-Lambert law (eq. e) is not valid over a broad concentration range. 

The protein-to-protein variation observed with the various protein assay methods makes it obvious why the largest source of error for protein assays is the choice of protein for the standard curve. If the sample contained IgG as the major protein and BSA was used for the standard curve, the estimated total protein concentration of the sample will be inaccurate. Whether the concentration was underestimated or overestimated depends upon which total protein assay method was used. If the Coomassie... 

Nucleic acids have substantial absorbance at 280 nm and can interfere with A2so quantitation of protein in crude samples. To resolve the protein concentration in such samples, measure the absorbance at 260 nm and 280 nm and calculate the protein concentration as follows (Warburg and Christian, 1942 Layne, 1957) protein concentration (mg/ml) = 1.55 x A2g0 -0.76 x A 260- This estimation of protein concentration is valid up to 20% (w/v) nucleic acid or an A2g )/A2(Jo ratio <0.6. 

ANS (see Basic Protocol 1) has been the most popular hydrophobic probe for the determination of surface hydrophobicity of proteins. Its dimeric form bis-ANS, which has a greater quantum yield in nonpolar environments by binding more strongly with proteins than the ANS monomer, is occasionally used for the same purpose (Das and Surewicz, 1995). These effects permit the observation of depolarization by energy transfer among the bound fluoropho-res, which can be used to estimate the distribution of the ligands among the protein molecules (Farris et al., 1978). 

The most popular method of electrophoretic separation by gels employs sodium dodecyl sulfate (SDS). This method not only gives an index of protein purity but yields an estimate of the protein subunit molecular weights. The mixture of proteins to be characterized is first completely denatured by adding SDS (a detergent) and mercaptoethanol and by briefly heating the mixture. Denaturation is caused by the association of the apolar tails of the SDS molecules with protein hydrophobic groups. Any cystine disulfide... 

Estimation of protein-bound iodine and tracer studies for the estimation of thyroid function are interfered with by the use of iodine-containing compounds (73). 

Cll. Crook, E. M., Harris, H., Hassan, F., and Warren, F. L., Continuous direct photometry of dyed materials in filter paper, with special reference to the estimation of proteins separated by electrophoresis. Biochem. J. 66, 434 (1954). 

In 1960, Hewitt and Notton estimated the phosphorus in fractions of tomato and cauliflower leaves using 32P coupled with extractions. Bowen et al. (1962) fed tomato plants with a number of radioactive elements, the fresh tissues were then extracted with a series of extractants. The results posed a number of interesting questions, for example, the association of cobalt and iron with proteins, and the authors suggested that a high speed centrifuge should be used to separate intracellular particles of different sizes. The nature of the metal compounds extracted was also questioned. Some inconclusive paper chromatography was also reported. 

Sreerama, N. and Woody, R.W. 2000. Estimation of protein secondary structure from circular dichroism spectra Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287(2) 252-260. 

While the redox titration method is potentiometric, the spectroelectrochemistry method is potentiostatic [99]. In this method, the protein solution is introduced into an optically transparent thin layer electrochemical cell. The potential of the transparent electrode is held constant until the ratio of the oxidized to reduced forms of the protein attains equilibrium, according to the Nemst equation. The oxidation-reduction state of the protein is determined by directly measuring the spectra through the tranparent electrode. In this method, as in the redox titration method, the spectral characterization of redox species is required. A series of potentials are sequentially potentiostated so that different oxidized/reduced ratios are obtained. The data is then adjusted to the Nemst equation in order to calculate the standard redox potential of the proteic species. Errors in redox potentials estimated with this method may be in the order of 3 mV. 

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